This project examines the hypothesis that reactive oxygen intermediates (ROI) overwhelm declining antioxidant defenses in aged RPE leading to mitochondrial DNA damage, cell dysfunction and death. This chain of events may be a primary mechanism of aging and of acquired retinal diseases such as AMD. A major objective of this study is to assess the basal levels, formation and repair of oxidative DNA damage in nuclear and mitochondrial DNA in primary RPE cells obtained from young versus aged human donors. We will test the hypothesis that mitochondrial DNA damage may accelerate the production of ROI that leads to further damage and functional decline. The following specific aims will be addressed to meet the objectives of this proposal: 1) To examine the formation and repair of oxidative DNA damage from rod outer segment-fed human retinal pigment epithelium (RPE) cell cultures; 2) To elucidate the temporal sequence and downstream cellular consequences in oxidant- exposed cultured human RPE cells in relation to mitochondrial DNA damage, mitochondrial transcription and function, and cell death; 3) To evaluate the linkage between reactive oxygen intermediates (ROI), antioxidant enzyme systems, and oxidant-induced DNA damage; 4) To study the relevance of the glutathione S-transferase pathway in protecting RPE cells under oxidative stress: a study with mGSTA4-4 transfected cells; 5) To examine the formation of mitochondrial DNA damage and repair kinetics in RPE from aged human eyes, and eyes with AMD. Successful accomplishment of these specific aims will provide fundamental mechanistic insights regarding the molecular pathobiology of reactive oxygen in aging RPE cells. The ultimate goal of this project is to reveal and test novel clinical strategies to reduce morbidity from age- related macular degeneration.